This application seeks to understand how the innate immune system influences the pathogenesis of Salmonella typhimurium. We are studying this issue by examining Salmonella virulence in mice and cells deficient in Toll- like receptors (TLRs). To unmask potentially critical host-pathogen interactions in this model, we have generated these TLR-deficient strains with a functional allele of Nramp-1, a divalent metal transporter that is mutated in many inbred mouse strains. Lack of functional Nramp-1 renders mice extremely susceptible to intracellular pathogens. Using these mouse strains we have made the surprising discovery that TLR- dependent phagosome acidification is required for induction of SPI-2 virulence genes. Thus, in macrophages lacking most or all TLR signaling, Salmonella is unable to create a replicative compartment. Surprisingly, this requirement for TLR signaling is only evident in cells with functional Nramp-1, suggesting that both TLRs and Nramp-1 manipulate the phagosomal environment. Salmonella is less virulent in mice lacking some, but not all, TLR signaling, consistent with an inability to replicate in macrophages. However, mice completely lacking TLR signaling are quite susceptible to Salmonella, suggesting that Salmonella is somehow able to cause disease in these animals without replicating in macrophages. The Aims of this grant focus on understanding how Nramp-1 and TLRs influence the niche where Salmonella replicates. In Aim 1, we will examine how Nramp-1 influences the phagosomal environment in the absence of TLR signaling. In Aim 2, we will examine how the level of TLR signaling can influence where Salmonella replicates in vivo. In Aim 3, we will exploit Salmonella's requirement for TLR signaling in mice with reduced TLR function. This requirement will be used to identify key cell types in which Salmonella must replicate while spreading systemically from intestinal sites.